ABSTRACT Alternative splicing of pre-mRNA enables a gene to give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. The long-term goal of our research is to understand the molecular mechanisms and function of alternative splicing on biological activities. The focus of this proposal is to investigate the regulation of alternative splicing in epithelial-mesenchymal transition (EMT). EMT is an essential developmental process that allows cells to change from a tightly packed cobble-stone-like epithelial cellular state to a motile and spindle-shaped mesenchymal cellular state. When abnormally activated, EMT promotes many types of diseases, including tissue fibrosis and cancer metastasis. Through working at the intersection of RNA splicing and cell biology, my lab has made several important findings connecting RNA regulation and EMT. Our work revealed, for the first time, that splice isoform switching of the CD44 gene causally controls EMT. By manipulating CD44 alternative splicing, we were able to convert cells between the epithelial and mesenchymal states. We have also identified the RNA-binding protein (RBP) hnRNPM as a critical splicing factor that promotes EMT through the regulation of alternative splicing. These results suggest that RNA splicing regulation could serve as an important mechanism that provides cellular plasticity. By shifting the programs of alternative splicing, cells are able to convert between the epithelial and mesenchymal cellular states. This capacity of reversing phenotypes is important for normal developmental EMT, as well as for cancer metastasis. Major new discoveries are necessary to fully understand this phenomenon and its underlying mechanisms. Our proposed research program is focused on (1) determining whether splice isoform switching acts as a prevalent mechanism that drives EMT or, if it is largely a phenomenon of byproducts; (2) understanding how RBPs precisely control alternative splicing during EMT; and (3) dissecting how signaling cascades elicit signals to RBPs and trigger alternative splicing changes during EMT. We have made major efforts in the past several years to build up experimental systems and gain expertise to help carry out our proposed studies. These efforts include large-scale RNA profiling of alternative splicing in multiple EMT systems, bioinformatics and experimental analysis of RBPs, as well as our recently completed kinase screen for EMT-associated alternative splicing alterations. Accomplishing the proposed work will provide new insights into our understanding of the regulatory mechanisms of alternative splicing, thus contributing to biological relevance in normal development and diseases.